Passive immunotherapy for influenza A H5N1 virus infection with equine hyperimmune globulin F(ab′)2 in mice

Background: Avian influenza virus H5N1 has demonstrated considerable pandemic potential. Currently, no effective vaccines for H5N1 infection are available, so passive immunotherapy may be an alternative strategy. To investigate the possible therapeutic effect of antibody against highly pathogenic H5N1 virus on a mammal host, we prepared specific equine anti-H5N1 IgGs from horses vaccinated with inactivated H5N1 virus, and then obtained the F(ab')(2) fragments by pepsin digestion of IgGs. Methods: The horses were vaccinated with inactivated H5N1 vaccine to prepare anti-H5N1 IgGs. The F(ab')(2) fragments were purified from anti-H5N1 hyperimmune sera by a protocol for 'enhanced pepsin digestion'. The protective effect of the F(ab')(2) fragments against H5N1 virus infection was determined in cultured MDCK cells by cytopathic effect (CPE) assay and in a BALB/c mouse model by survival rate assay. Results: By the protocol for 'enhanced pepsin digestion', total 16 g F(ab')(2) fragments were finally obtained from one liter equine antisera with the purity of over 90%. The H5N1-specific F(ab')(2) fragments had a HI titer of 1: 1024, and the neutralization titre of F(ab')(2) reached 1: 2048. The in vivo assay showed that 100 mu g of the F(ab')(2) fragments could protect BALB/c mice infected with a lethal dose of influenza H5N1 virus. Conclusion: The availability of highly purified H5N1-specific F(ab')(2) fragments may be promising for treatment of influenza H5N1 infection. Our work has provided experimental support for the application of the therapeutic equine immunoglobulin in future large primate or human trials.